摘要
目的:观察骨髓内脂肪细胞对骨髓基质细胞成骨能力的影响,以及能否产生诱导骨髓基质细胞凋亡的作用。方法:实验于2001-07/2003-06在华中科技大学同济医学院附属同济医院骨科实验室完成。将骨髓基质细胞以109L-1的浓度接种于培养板和培养瓶中,加入含体积分数为0.1的胎牛血清,100U/mL青霉素,100mg/L链霉素的DMEM培养基。上述细胞随机分为空白对照组、106,107,108,109L-1脂肪细胞组,分别加入0,106,107,108,109L-1不同浓度的脂肪细胞,37℃、体积分数为0.05的CO2饱和湿度培养箱中培养,建立脂肪细胞和骨髓基质细胞共培养体系。培养时间为12d,每4d取各组细胞样本检测细胞内碱性磷酸酶活性,利用原位杂交方法检测I型胶原mRNA表达,3H-脯氨酸掺入实验检测骨髓基质细胞胶原合成能力,并以TUNEL法及流式细胞仪检测细胞凋亡。结果:①共培养体系建立后细胞形态学观察:细胞共培养体系建立48h后,细胞开始贴壁。72h后贴壁的骨髓基质细胞主要为短梭形,部分为多边形,并伸出突起,体积增大,具有典型的成纤维细胞特点。贴壁的脂肪细胞呈多角形或圆形,细胞透明度较好。②脂肪细胞对骨髓基质细胞碱性磷酸酶活性的影响:与空白对照组比较,培养第4,8,12天随着共培养体系中脂肪细胞的浓度不断上升,骨髓基质细胞内碱性磷酸酶活性均有不同程度的下降(P<0.05或P<0.01)。③脂肪细胞对骨髓基质细胞内I型胶原mRNA表达的影响:空白对照组染色最深,即表达最强,随着培养体系中脂肪细胞浓度增加,染色由深逐渐变浅,即I型胶原mRNA表达逐渐减弱。④3H-脯氨酸掺入实验结果:空白对照组第4天每分钟脉冲数值最高,随时间延长有所下降。随脂肪细胞浓度的上升,各组每分钟脉冲数值均有不同程度降低,并具有浓度及时间依赖的特征。⑤细胞凋亡TUNEL检测结果:在含有脂肪细胞的共培养体系中,被标记呈棕色的凋亡细胞,细胞核染色质浓聚成致密的斑点状,核固缩甚至碎裂。同一时间点106,107,108,109L-1脂肪细胞组凋亡细胞明显多于空白对照组(P<0.01),空白对照组中几乎没有凋亡细胞。⑥细胞凋亡流式细胞仪检测结果:各组悬浮细胞经流式细胞仪检测后,空白对照组细胞均集中于左下象限,共培养体系中均可在右下象限发现有凋亡细胞存在。结论:髓内脂肪细胞干扰了骨髓基质细胞的成骨能力,并能够诱导骨髓基质细胞的凋亡,可能与原发性骨质疏松症的发病有关。
AIM： To observe the effects of adipocyte in bone marrow cavity on osteogenic potential of bone marrow stromal cells, and if adipocyte could induce apoptosis of bone marrow stromal cell. METHODS： This experiment was conducted at the laboratory of Department of Orthopaedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technoloby from July 2001 to June 2003. Bone marrow stromal cells were inoculated in culture plate and Petri dish with the concentration Of 10^9/L, DMEM culture medium containing 0.1 volume fraction of fetal bovine serum, 100 U/mL penicillinum and 100 mg/L streptomycin was added. The above-mentioned cells were randomly divided into blank control group, l0^6, 10^7, l0^5, l0^9 L^-l adipocytes groups , in which some adipocytes with concentration of 0, l0^6, l0^7, l0^8, 10^9/L were added, respectively. All the cells were cultured in the saturated humidity culture box with 0.05 volume fraction of CO2 at 37℃. The co-culture system of adipocyte and bone marrow stromal cells was established. After 4, 8, 12 days, alkaline phosphatase （ALP） activities of the samples were detected, the mRNA expression of collagen type I was detected with in situ hybridization method, the collagen synthesis ability with 3^H-proline in corporation test. Cell apoptosis was detected with terminal deoxynucleotidyl transferase-mediated dUTP mick end- labeling （TUNEL） and flow cytometer.
RESULTS： ① Cellular morphological observation after establishing coculture system： After 48 hours, cells began to adhere to the wall. 72 hours later, the adhered bone marrow stromal cells mainly presented short-shuttle shape, some were polygon and mutations stretched out, and the Volume was enlarged, possessing typical characteristics of fibroblasts. The adhered adipocytes presented polygon or round shape and good cellular transparence. ② Effect of adipocytes on ALP activities of bone marrow stromal cells： as compared with blank control group, with the increase of concentration of adipocytes in the co-culture system, ALP activities of bone marrow stromal cells all had decrease at different degrees at the 4th, 8th and 12th days of the culture （P 〈0.05 or P〈0.01 ）.③ Effect of adipocytcs on mBNA expression of collagen type I in bone marrow stromal cells： The. staining was the darkest in blank control group that was to say, the expression was the strongest. With the increase of adipocytes in the co-culture system, the staining gradually became from dark to light, that was to say, collagen I mRNA expression was gradually weakened. ④ 3^H-proline in corporation test, in blank control group, counts per minute was increased on the 4th day . With the elongation of time, counts per minute had a little decrease. With the increase of concentration of adipocytes, counts per minute had decrease atdifferent degrees in each group with concentration-time dependent manner. ⑤ TUNEL detection of apoptotie cells： In the co-culture system containing adipoeytes, nuclear ehromatin of brown-labeled apoptotie cells condensed into dense spot shape, karyopyenosis even eataelasm appeared. At the same time, apoptotie cells were significantly more in the l0^6, 10^7,10^8, 10^9 L^-1 adipoeytes group than in blank control group （P〈0.01）, there was seldomly apoptotie cells in blank control group. ⑥ Detection result of cellular apoptosis by flow eytometer ： Through detecting suspended cells by flow eytometer： in blank control group, cells gathered left down quadrant, apoptotie cells of co-culture system could be found in the right down quadrant.
CONCLUSION： Adipocytes in bone marrow eavity interfere osteogenic potential of bone marrow stromal cell, and induce apoptosis of bone marrow stromal cell. It might be related to the morbidity of primary osteoporosis.
出处
《中国临床康复》
CSCD
北大核心
2006年第29期94-97,i0004,共5页
Chinese Journal of Clinical Rehabilitation
